CN105067817A - Methods and devices for acquiring signals and tracking cells by adopting light sensitive chips - Google Patents

Methods and devices for acquiring signals and tracking cells by adopting light sensitive chips Download PDF

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Publication number
CN105067817A
CN105067817A CN201510398509.0A CN201510398509A CN105067817A CN 105067817 A CN105067817 A CN 105067817A CN 201510398509 A CN201510398509 A CN 201510398509A CN 105067817 A CN105067817 A CN 105067817A
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sensitive chip
signal
darkroom
cell
light
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CN105067817B (en
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张英豪
詹克团
宛佳
张志豪
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Shanghai Yi Bei Photoelectric Technology Co. Ltd.
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Shanghai Qingliu Biological Pharmaceutical Co Ltd
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Priority to CN201510398509.0A priority Critical patent/CN105067817B/en
Publication of CN105067817A publication Critical patent/CN105067817A/en
Priority to PCT/CN2016/084913 priority patent/WO2017005075A1/en
Priority to CA2993873A priority patent/CA2993873C/en
Priority to KR1020187003230A priority patent/KR102084233B1/en
Priority to RU2018104813A priority patent/RU2715228C2/en
Priority to JP2018520007A priority patent/JP2018524613A/en
Priority to EP16820726.4A priority patent/EP3315971A4/en
Priority to AU2016289902A priority patent/AU2016289902B2/en
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Publication of CN105067817B publication Critical patent/CN105067817B/en
Priority to US15/864,170 priority patent/US20180136219A1/en
Priority to JP2020067922A priority patent/JP2020115149A/en
Priority to US17/674,836 priority patent/US20220260499A1/en
Priority to JP2022125453A priority patent/JP2022140791A/en
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76Chemiluminescence; Bioluminescence
    • G01N21/763Bioluminescence
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
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    • C09KMATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/61Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing fluorine, chlorine, bromine, iodine or unspecified halogen elements
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • GPHYSICS
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    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76Chemiluminescence; Bioluminescence
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531Production of immunochemical test materials
    • G01N33/532Production of labelled immunochemicals
    • G01N33/535Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G01N33/561Immunoelectrophoresis
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40Concentrating samples
    • G01N2001/4038Concentrating samples electric methods, e.g. electromigration, electrophoresis, ionisation
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N2021/1765Method using an image detector and processing of image signal

Abstract

The invention discloses methods and devices for acquiring signals and tracking cells by adopting light sensitive chips, and relates to the technical field of signal acquisition. The signal acquiring method adopting the light sensitive chip comprises the following steps: a light-emitting surface carrying a to-be-acquired light signal film is tightly attached to the light sensitive chip, the light sensitive chip attached with the to-be-acquired light signal film is placed in a darkroom, and the light sensitive chip acquires light signals in the darkroom, processes the acquired light signals and outputs the signals. The signal acquiring method and device adopting the light sensitive chip acquire the signals by the aid of contact with the light sensitive chip, the light signals are converted into digital signals, and quantitative analysis is finished very well.

Description

The method and apparatus of sensitive chip collection signal and the method and apparatus of tracking cell
Technical field
The present invention relates to information acquiring technology field, particularly relate to a kind of method and apparatus of sensitive chip collection signal and follow the trail of the method and apparatus of cell.
Background technology
Western blot (Westernblotting) is a kind of hybridization technique high resolving power gel electrophoresis and immunochemical analyses technology combined.Western blot has the advantages such as analysis capacity is large, susceptibility is high, high specificity, detect protein properties, express and a kind of the most frequently used method distributed, as the antibody of the qualitative and quantitative detection of tissue antigen, the quality determination of peptide molecule and virus or antigen detection etc.
The apparatus and method of existing collection Western blot (Westernblotting) signal:
Method one: sensitive film and NC film are close together, after exposure certain hour, carries out developing, fixing.Image is presented on film.Advantage: highly sensitive, resolution is high.There is following shortcoming: 1, take up room large: need special darkroom (room), tank, and sewer pipe etc.2, spend high: not only need to buy developing machine, camera obscura, also needs the consumptive materials such as a large amount of sensitive film, developer solution, stop bath.Because of washing film, also water resource waste can be caused.3, contaminated environment: develop photographic film and need a large amount of developer solution, stop bath.Produce because of film off quality and discarded simultaneously.These can cause the pollution such as heavy metal and aromatic.4, picture quality is unstable: in darkroom, and researchist cannot Real-Time Monitoring depth of exposure, often attempts just obtaining good image through for several times.Be not under-exposure time most be exactly over-exposed.Waste time and energy.5, time-consuming: nowadays data are preserved, transmitted and deliver and generally adopt digitizing, and thus, film image also will become digital picture through scan transformation.6, quantitatively inaccurate: Many times, researchist's naked eyes judge to think good image, in fact supersaturation in gray scale.Thus follow-up gray scale scanning is difficult to accurate quantitative analysis.
Method two: utilize the lighting apparatus such as CCD directly to take a picture to sample.Advantage: all shortcomings overcoming method one.Shortcoming: lost the advantage that film gathers, namely sensitivity seriously reduces.Reason is: on Vehicles Collected from Market, this kind equipments all are all by CCD digital camera frame shooting image in a distance above NC film.The light of luminous energy only in a certain low-angle of radiation of light source could by collected by camera.The energy of more than 90% runs off.Thus this kind equipment is by through being usually used in intense light irradiation phase.That only has individual brand claims the low light level photography that can be used for WB.But compared with film, its time shutter extends greatly.Some brand adopts the method for potting gum, sacrifices resolution to improve sensitivity, can reach the sensitivity be equal to film.But image amplifies a little, just present mosaic, be difficult to meet many-sided demand.
Method three: utilize linearly aligned CCD photosensitive unit, low level light signal is collected in scanning.Advantage: improve the acquisition rate to light signal, thus improve sensitivity.Shortcoming: owing to being that light signal is collected in linear sweep, can not gather the image of view picture, thus zones of different scanning life period is poor simultaneously.Again because light source is constantly decay in time, the signal intensity that different time points is collected thus can be caused there is no comparability.A lot of control test cannot be compared.
Therefore, the technical matters needing those skilled in the art urgently to solve at present is exactly: how can innovatively propose a kind of effective measures, to solve problems of the prior art, to meet the more demands in practical application.
Summary of the invention
Embodiment of the present invention technical matters to be solved is to provide a kind of method of sensitive chip collection signal and follows the trail of the method for cell, measured signal is converted into digital signal by light signal, realizes quantitative test fast.
Accordingly, the embodiment of the present invention additionally provides a kind of device of sensitive chip collection signal and follows the trail of the device of cell, in order to ensure the implementation and application of said method.
In order to solve the problem, the invention discloses a kind of method of sensitive chip collection signal, comprising:
The light-emitting area of carrying light signal film to be collected fits tightly on sensitive chip;
Darkroom is placed in by being fitted with the sensitive chip carrying light signal film to be collected; Described darkroom does not affect by ambient light;
In described darkroom, sensitive chip carries out light signal collection;
Signal transacting is carried out to gathered light signal and exports.
Preferably, carry out light signal collection at described darkroom sensitive chip to comprise the following steps:
Collect light signal;
By computer screen Real Time Observation depth of exposure;
Exposure is stopped when signal accumulation arrives preset strength;
Obtain and preserve the image exposing and produce.
Preferably, when described sensitive chip collection signal is Western blotting signal, described in carry light signal film to be collected acquisition comprise the following steps:
First testing protein leakage of electricity is swum;
The gel running through electrophoresis carries out protein delivery;
Testing protein in gel is transferred to PVDF membrane or nitrocellulose filter;
The PVDF membrane shifted or nitrocellulose filter are closed, adds the primary antibodie reaction of anti-testing protein, add two anti-HRP reactions;
Chemical luminescence for liquid process is carried out to reacted PVDF membrane or nitrocellulose filter.
Preferably, described film comprise nitrocellulose filter and or PVDF membrane.
Preferably, described sensitive chip comprises CMOS sensitive chip and CCD sensitive chip.
The invention also discloses a kind of device of sensitive chip collection signal, comprising:
Laminating module, for fitting tightly at sensitive chip by the light-emitting area of carrying light signal film to be collected;
Shelve module, for being placed in darkroom by being fitted with the sensitive chip carrying light signal film to be collected; Described darkroom does not affect by ambient light;
Signal acquisition module, carries out light signal collection for completing at described darkroom sensitive chip;
Signal processing module, for carrying out signal transacting to gathered light signal and exporting.
The invention also discloses a kind of method that sensitive chip follows the trail of cell, comprising:
The cell or the animal that carry luciferase are planted on sensitive chip;
Darkroom is placed in by planting the sensitive chip being implanted with the cell or animal carrying luciferase; Described darkroom does not affect by ambient light;
In described darkroom, sensitive chip carries out light signal collection;
Signal transacting is carried out to gathered light signal and exports.
Preferably, described method also comprised before being planted on sensitive chip by the cell or animal that carry luciferase: equipped with glass layer on sensitive chip, and then the cell or animal that carry luciferase was planted in the glassy layer on sensitive chip.
Preferably, the acquisition of the cell or animal that carry luciferase described in comprises the following steps:
Build the reporter plasmid that the specific fragment of target promoter is inserted into luciferase expression sequence front by;
By in the embryonated egg of regulating and controlling sequence and luciferase gene plasmid co-transfection cell or animal;
Fluorescein is added in cell culture medium.
The invention also discloses the device that a kind of sensitive chip follows the trail of cell, comprising:
Plantation module, for being planted in sensitive chip by the cell or animal that carry luciferase;
Shelve module, for being placed in darkroom by planting the sensitive chip being implanted with the cell or animal carrying luciferase; Described darkroom does not affect by ambient light;
Signal acquisition module, carries out light signal collection for completing at described darkroom sensitive chip;
Signal processing module, for carrying out signal transacting to gathered light signal and exporting.
Compared with prior art, the embodiment of the present invention comprises following advantage:
Scheme provided by the invention is by fitting tightly on sensitive chip by the light-emitting area of carrying light signal film to be collected, darkroom is placed in by being fitted with the sensitive chip carrying light signal film to be collected, in described darkroom, sensitive chip carries out light signal collection, and carries out signal transacting to gathered light signal and export.Direct contact sensitive chip settling signal gathers, and can gather simultaneously, farthest avoid loss of optical signal, thus carrying highly sensitive while, do not reduce resolution to the image of view picture.So not only remain all advantages of three kinds of methods in background technology, it also avoid their respective shortcomings simultaneously.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, accompanying drawing in the following describes is some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is the method flow schematic diagram of a kind of sensitive chip collection signal of the present invention;
Fig. 2 is the apparatus structure schematic diagram of a kind of sensitive chip collection signal of the present invention;
Fig. 3 is the method flow schematic diagram of a kind of sensitive chip track cells of the present invention;
Fig. 4 is the apparatus structure schematic diagram of a kind of sensitive chip track cells of the present invention.
Embodiment
For making the object of the embodiment of the present invention, technical scheme and advantage clearly, below in conjunction with the accompanying drawing in the embodiment of the present invention, technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment one
Introduce the method for a kind of sensitive chip collection signal that the embodiment of the present invention provides in detail.
With reference to Fig. 1, show the schematic flow sheet of the embodiment of the method for a kind of sensitive chip collection signal of the present invention, specifically can comprise the steps:
Step 101, the light-emitting area of carrying light signal film to be collected fits tightly on sensitive chip;
In actual applications, when use sense optical chip gathers Western blotting signal, the acquisition of carrying light signal film to be collected comprises the following steps:
First testing protein leakage of electricity is swum;
The gel running through electrophoresis carries out protein delivery;
Testing protein in gel is transferred to PVDF membrane or nitrocellulose filter;
The PVDF membrane shifted or nitrocellulose filter are closed, adds the primary antibodie reaction of anti-testing protein, add two anti-HRP reactions;
Reacted PVDF membrane or nitrocellulose filter are carried out chemical luminescence for liquid process.
In application, the film used mainly comprise nitrocellulose filter (NC film) and or PVDF membrane (pvdf membrane).The sensitive chip used comprises CMOS sensitive chip and CCD sensitive chip etc.Consider that the sensitive chip manufacturing cost of large-size is high, if utilize chip splicing can arrive good effect in practice, cost will reduce greatly, therefore also can be the array of CMOS sensitive chip or CCD sensitive chip.
Step 102, is placed in darkroom by being fitted with the sensitive chip carrying light signal film to be collected; Described darkroom does not affect by ambient light;
In order to the impact not by ambient light, need be placed in dark surrounds by being fitted with the sensitive chip carrying light signal film to be collected, implementation can be selected according to specific environment, realizes than being easier to, can to the individual lid that is in the light of its button.
Step 103, in described darkroom, sensitive chip carries out light signal collection;
In actual application, carry out light signal collection at described darkroom sensitive chip and comprise the following steps:
Collect light signal;
By computer screen Real Time Observation depth of exposure;
Exposure is stopped when signal accumulation arrives preset strength;
Obtain and preserve the image exposing and produce.
Step 104, carries out signal transacting to gathered light signal and exports.
Single-chip microcomputer can be adopted in application, the processor of FPGA, CPU etc. carries out signal transacting to gathered light signal and exported.Method compare based on current signal transacting is ripe, and can complete the signal transacting described in this programme, therefore repeats no more here.
Gather Western blotting (WB signal) below in conjunction with practical application to sensitive chip to describe in detail:
Western blotting principle of work:
1, electric field is utilized longitudinally to be separated on polyacrylate hydrogel by cell or tissue extract (as left figure, figure below is step ground flow process for this reason);
2, utilize electric field by protein band horizontal transfer on nitrocellulose filter (NC film), band relative position does not change, and is called trace (Blot);
3, close.Film after trace is soaked in bovine serum albumin(BSA) (BSA) solution.The region making BSA occupy protein band not occupy.Avoid follow-up antibody to be adsorbed by these regions, make antibody can only combination single-minded with its antigen.
4, antibody incubation.Antibody in advance and horseradish peroxidase (HRP) hinge.Antibody is soaked together with NC film and hatches.Antibody is combined with its antigen-specific.
5, signals collecting.NC film is immersed in the liquid containing HRP substrate.Fluorescence is discharged during its substrate of HRP catalysis.Photosensitive film or sense electronics photosystem is utilized to gather fluorescence signal.
Step before WB light signal collection
(1) protein example obtains: after induced expression in bacterium, by electrophoresis sample-loading buffer By Direct Pyrolysis cell, eukaryotic adds homogenate buffer, machinery or ultrasound wave room temperature homogenate 0.5-1min.Then 4 DEG C, the centrifugal 15min of 13,000g.Get supernatant as sample.
(2) electrophoresis: PAGE production gel, carries out SDS-PAGE.
(3) shift: 1. adhesive tape is cut to suitable size after terminating by electrophoresis, by transferring film damping fluid balance, 5min × 3 time.2. film process: cut out the filter paper onesize with adhesive tape and NC film in advance, immerses 10min in transferring film damping fluid.3. transferring film: membrane-transferring device is put well by the order of carbon anode plate, 24 metafiltration paper, NC film, gel, 24 metafiltration paper, negative electrode carbon plate from bottom to up successively, filter paper, gel, NC film Accurate align, each step removes bubble, and upper pressure 500g weight, blots liquid unnecessary on carbon plate.Switch on power, constant current 1mA/cm2, transfer 1.5hr.After transfer terminates, film takes out by deenergization, extracts film bar to be measured and does Western blotting.By there being the band of protein standard to dye, after putting into film dyeing liquor 50s, repeatedly decolour in 50% methyl alcohol, to clear background, then wash with distilled water, air-dry being sandwiched in two layers of filter paper is preserved, and stays and compares with the result that develops the color.
(4) immune response: wash film with 0.01MPBST, 5min × 3 time.
Add confining liquid, steadily shake, room temperature 1hr.
Abandon coating buffer, wash film with 0.01MPBST, 5min × 3 time.
Add primary antibodie (dilute in appropriate dilutions ratio 0.01MPBS, liquid must coverlay whole), place more than 12hr for 4 DEG C.Negative control, replace primary antibodie with 1%BSA, all the other steps are identical with experimental group.
Abandon primary antibodie and 1%BSA, wash film respectively with 0.01MPBS, 5min × 4 time.
Add two anti-(diluting in appropriate dilutions ratio 0.01MPBST) of horseradish peroxidase, steadily shake, room temperature 2hr.
Abandon two to resist, wash film with 0.01MPBST, 5min × 4 time.
Chemiluminescence liquid treatment membrane, HRP meets the chemical substrate in liquid, and catalytic substrate also sends fluorescence.
Utilize digital photosensitive chip directly and the film method of fitting gather fluorescence signal:
The film be immersed in chemical luminescence for liquid is taken out, thieving paper drains surplus liquid, then the light-emitting area of film is attached on digital photosensitive chip;
With smooth object press mold, itself and digital photosensitive chip are directly fitted tightly;
Buckle lid, make digital photosensitive chip and film be in dark surrounds, avoid ambient light to pollute;
Control sensitive chip by computing machine, start to collect chemiluminescent signal.By computer screen Real Time Observation depth of exposure, when signal accumulation arrives suitable intensity, stop exposure;
Obtain and preserve the image exposing and produce, image can be used for quantitatively and qualitative analysis.
In the present embodiment, by the collection of contact sensitive chip settling signal, by light signal is converted into digital signal, can be good at quantitative test.
Embodiment two
Introduce the device of a kind of sensitive chip collection signal that the embodiment of the present invention provides in detail.
See Fig. 2, show the apparatus structure schematic diagram of a kind of sensitive chip collection signal of the present invention, specifically comprise:
Laminating module 201, for fitting tightly at sensitive chip by the light-emitting area of carrying light signal film to be collected;
Shelve module 202, for being placed in darkroom by being fitted with the sensitive chip carrying light signal film to be collected; Described darkroom does not affect by ambient light;
Signal acquisition module 203, carries out light signal collection for completing at described darkroom sensitive chip;
Signal processing module 204, for carrying out signal transacting to gathered light signal and exporting.
For device embodiment, due to itself and embodiment of the method basic simlarity, so description is fairly simple, relevant part illustrates see the part of embodiment of the method.
Embodiment three
A kind of sensitive chip that introducing the embodiment of the present invention in detail provides follows the trail of the method for cell.
With reference to Fig. 3, show the method that a kind of sensitive chip of the present invention follows the trail of cell, specifically can comprise:
Step 301, the cell or the animal that carry luciferase are planted on sensitive chip;
Sensitive chip can cover usually the protective seam of layer of transparent, or glassy layer or resin bed are again or other materials etc., the thickness of protective seam is less than 0.5mm usually.
Consider in practice that the secondary following the trail of effect and sensitive chip uses, before the cell or animal that carry luciferase are planted on sensitive chip can on sensitive chip equipped with glass layer, and then the cell or animal that carry luciferase are planted in the glassy layer of sensitive chip, like this can be convenient in the cleaning in later stage.
Not there is for employing sensitive chip the instrument of light emitting module, usually manage to allow cell or meiofauna (as nematode and fruit bat etc.) etc. possess autoluminescence ability, the concrete luciferase gene that proceeds to makes cell or animals and plants generation autoluminescence ability be the ways relatively commonly used.
Luciferase (English name: Luciferase) is a kind of albumen that firefly afterbody produces, and can deposit at ATP in case can catalytic fluorometry element (luciferin) and oxygen reaction, sends fluorescence.Utilize bionic way, the gene of luciferase is proceeded in cell or animal and plant body together with the DNA sequence dna being used for regulatory transcription, and is incorporated on the chromosome of host.Some utilizing host expresses has the protein molecule of special construction, exerts regulatory gene expression function, specifically in conjunction with the DNA sequence dna of regulatory transcription, thus plays enhancing to the expression of luciferase genes.
The acquisition of the described cell or animal that carry luciferase comprises the following steps:
Build the reporter plasmid that the specific fragment of target promoter is inserted into luciferase expression sequence front by; Concrete as pGL3-basic etc.;
By in the embryonated egg of regulating and controlling sequence and luciferase gene plasmid co-transfection cell or animal (transgenic animals);
In cell culture medium, add fluorescein, luciferase utilizes intracellular ATP to provide energy catalysis fluorescein and oxygen reaction, produces fluorescence.
Like this, instrument can follow the trail of the traveling locus of cell or animal.The method can be used for Animal Behavior Science experiment.
Step 302, is placed in darkroom by planting the sensitive chip being implanted with the cell or animal carrying luciferase; Described darkroom does not affect by ambient light;
Step 303, in described darkroom, sensitive chip carries out light signal collection;
Step 304, carries out signal transacting to gathered light signal and exports.
Embodiment four
A kind of sensitive chip that introducing the embodiment of the present invention in detail provides follows the trail of the device of cell.
See Fig. 4, show the apparatus structure schematic diagram that a kind of sensitive chip of the present invention follows the trail of cell, specifically comprise:
Plantation module 401, for being planted in sensitive chip by the cell or animal that carry luciferase;
Shelve module 402, for being placed in darkroom by planting the sensitive chip being implanted with the cell or animal carrying luciferase; Described darkroom does not affect by ambient light;
Signal acquisition module 403, carries out light signal collection for completing at described darkroom sensitive chip;
Signal processing module 404, for carrying out signal transacting to gathered light signal and exporting.
Scheme of the present invention can be widely used in gathering Western blotting signal, monitoring rate compared with the scotopic power in droplet array, repopulating cell observation of cell migration on sensitive chip, division, or the dynamic process of some developed by molecule etc.
It should be noted that, for embodiment of the method, in order to simple description, therefore it is all expressed as a series of combination of actions, but those skilled in the art should know, the embodiment of the present invention is not by the restriction of described sequence of movement, because according to the embodiment of the present invention, some step can adopt other orders or carry out simultaneously.Secondly, those skilled in the art also should know, the embodiment described in instructions all belongs to preferred embodiment, and involved action might not be that the embodiment of the present invention is necessary.
For device embodiment, due to itself and embodiment of the method basic simlarity, so description is fairly simple, relevant part illustrates see the part of embodiment of the method.
Each embodiment in this instructions all adopts the mode of going forward one by one to describe, and what each embodiment stressed is the difference with other embodiments, between each embodiment identical similar part mutually see.
Above to the method and apparatus of a kind of sensitive chip collection signal provided by the present invention and the method and apparatus of tracking cell, be described in detail, apply specific case herein to set forth principle of the present invention and embodiment, the explanation of above embodiment just understands method of the present invention and core concept thereof for helping; Meanwhile, for one of ordinary skill in the art, according to thought of the present invention, all will change in specific embodiments and applications, in sum, this description should not be construed as limitation of the present invention.

Claims (10)

1. a method for sensitive chip collection signal, is characterized in that, comprising:
The light-emitting area of carrying light signal film to be collected fits tightly on sensitive chip;
Darkroom is placed in by being fitted with the sensitive chip carrying light signal film to be collected; Described darkroom does not affect by ambient light;
In described darkroom, sensitive chip carries out light signal collection;
Signal transacting is carried out to gathered light signal and exports.
2. the method for claim 1, is characterized in that, carries out light signal collection comprise the following steps at described darkroom sensitive chip:
Collect light signal;
By computer screen Real Time Observation depth of exposure;
Exposure is stopped when signal accumulation arrives preset strength;
Obtain and preserve the image exposing and produce.
3. the method for claim 1, is characterized in that, when described sensitive chip collection signal is Western blotting signal, described in carry light signal film to be collected acquisition comprise the following steps:
First testing protein leakage of electricity is swum;
The gel running through electrophoresis carries out protein delivery;
Testing protein in gel is transferred to PVDF membrane or nitrocellulose filter;
The PVDF membrane shifted or nitrocellulose filter are closed, adds the primary antibodie reaction of anti-testing protein, add two anti-HRP reactions;
Chemical luminescence for liquid process is carried out to reacted PVDF membrane or nitrocellulose filter.
4. the method for claim 1, is characterized in that, described film comprise nitrocellulose filter and or PVDF membrane.
5. the method for claim 1, is characterized in that, described sensitive chip comprises CMOS sensitive chip and CCD sensitive chip.
6. a device for sensitive chip collection signal, is characterized in that, comprising:
Laminating module, for fitting tightly at sensitive chip by the light-emitting area of carrying light signal film to be collected;
Shelve module, for being placed in darkroom by being fitted with the sensitive chip carrying light signal film to be collected; Described darkroom does not affect by ambient light;
Signal acquisition module, carries out light signal collection for completing at described darkroom sensitive chip;
Signal processing module, for carrying out signal transacting to gathered light signal and exporting.
7. sensitive chip follows the trail of a method for cell, it is characterized in that, comprising:
The cell or the animal that carry luciferase are planted on sensitive chip;
Darkroom is placed in by planting the sensitive chip being implanted with the cell or animal carrying luciferase; Described darkroom does not affect by ambient light;
In described darkroom, sensitive chip carries out light signal collection;
Signal transacting is carried out to gathered light signal and exports.
8. method as claimed in claim 7, it is characterized in that, described method also comprised before being planted on sensitive chip by the cell or animal that carry luciferase: equipped with glass layer on sensitive chip, and then the cell or animal that carry luciferase was planted in the glassy layer on sensitive chip.
9. as claimed in claim 7 or 8 method, is characterized in that, described in carry luciferase the acquisition of cell or animal comprise the following steps:
Build the reporter plasmid that the specific fragment of target promoter is inserted into luciferase expression sequence front by;
By in the embryonated egg of regulating and controlling sequence and luciferase gene plasmid co-transfection cell or animal;
Fluorescein is added in cell culture medium.
10. sensitive chip follows the trail of a device for cell, it is characterized in that, comprising:
Plantation module, for being planted in sensitive chip by the cell or animal that carry luciferase;
Shelve module, for being placed in darkroom by planting the sensitive chip being implanted with the cell or animal carrying luciferase; Described darkroom does not affect by ambient light;
Signal acquisition module, carries out light signal collection for completing at described darkroom sensitive chip;
Signal processing module, for carrying out signal transacting to gathered light signal and exporting.
CN201510398509.0A 2015-07-08 2015-07-08 Methods and devices for acquiring signals and tracking cells by adopting light sensitive chips Expired - Fee Related CN105067817B (en)

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CN201510398509.0A CN105067817B (en) 2015-07-08 2015-07-08 Methods and devices for acquiring signals and tracking cells by adopting light sensitive chips
JP2018520007A JP2018524613A (en) 2015-07-08 2016-06-06 Method and apparatus for signal collection by photosensitive chip, and method and apparatus for cell tracking
AU2016289902A AU2016289902B2 (en) 2015-07-08 2016-06-06 Method and apparatus for collecting signal, and method and apparatus for tracking cell by using light sensitive chip
CA2993873A CA2993873C (en) 2015-07-08 2016-06-06 Method and apparatus for collecting signals, and method and apparatus for tracking cells by using light sensitive chip
KR1020187003230A KR102084233B1 (en) 2015-07-08 2016-06-06 Signal acquisition method and device of photosensitive chip and cell tracking method and device
RU2018104813A RU2715228C2 (en) 2015-07-08 2016-06-06 Method and apparatus for collecting signals and method and apparatus for tracking cells using photosensitive microcircuit
PCT/CN2016/084913 WO2017005075A1 (en) 2015-07-08 2016-06-06 Method and apparatus for collecting signal, and method and apparatus for tracking cell by using light sensitive chip
EP16820726.4A EP3315971A4 (en) 2015-07-08 2016-06-06 Method and apparatus for collecting signal, and method and apparatus for tracking cell by using light sensitive chip
US15/864,170 US20180136219A1 (en) 2015-07-08 2018-01-08 Method and apparatus for collecting signal, and method and apparatus for tracking cell by using light sensitive chip
JP2020067922A JP2020115149A (en) 2015-07-08 2020-04-03 Method and device for collecting signal using photosensitive chip, and method and device for tracking cell
US17/674,836 US20220260499A1 (en) 2015-07-08 2022-02-17 Method and apparatus for collecting signals, tracking cells, and imaging control using a photosensitive chip
JP2022125453A JP2022140791A (en) 2015-07-08 2022-08-05 Method and device for collecting signal using photosensitive chip, and method and device for tracking cell

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